The major histocompatibility complex (MHC) is a set of linked genes which code for cell surface proteins involved in transplant rejection. The MHC contains three types of genes, class I, II and III (Klein J. et al.: Immunology: The Science of Self-Nonself Discrimination, pp. 687, 1984, John Wiley, Somerset, N.J.).
In humans, class I genes encode polymorphic 44,000 dalton glycoprotein chains that associate with a nonpolymorphic 12,000 dalton light chain, xcex22-microglobulin, and which are expressed on most cells of the body. Typical class I MHC genes are involved in regulating immune to viral infections (Zinkemagal R. M. et al. (1979) Adv. Immunol. 27:52-72).
In humans, the class II MHC antigens are cell surface glycoproteins composed of an xcex1 chain of approximately 35,000 daltons and a xcex2 chain of about 28,000 that are expressed only on subsets of immunologically active cells, such as xcex2 lymphocytes and macrophages.
Class III MHC genes code for serum proteins such as complement (Cxe2x80x2).
The MHC loci in swine are known as the swine leukocyte antigens (SLA). In 1970, Vaiman et al., (Vaiman M. et al. (1970) Transplantation 10: 155-161) and Viza et al. (Viza D. et al. (1970) Nature 227:949-951) provided descriptions of the SLA complex. These groups developed panels of SLA typing reagents (Vaiman M. et al. (1979) Immunogenetics 9:353-361) by preparing antisera of defined specificity as well as by characterizing cells of known SLA type (homozygous typing cells) for use in mixed lymphocyte complex, to chromosome 7 (Geffrotin C. et al. (1984) Ann Genet (Parix) 27:213-219). The class I swine MHC loci are designated SLA-A,B,C. The class II swine MHC loci are designated SLA-DR, DQ. Because there are numerous genes coded by the SLA complex and because usually they are inherited as a unit, haplotype designations have been developed. For example, the SLAa haplotype codes for SLA-AaBaCaDRaDQa alleles.
Miniature swine are a good model for organ transplantation studies because of their breeding characteristics which make them one of few large animals in which genetics can be manipulated in a reasonable time, and also because of their size which permits surgical manipulations similar to those humans.
The invention provides a genetically defined, large animal, useful, e.g., as an organ, tissue, or cell, donor, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and preferably in which a sufficient number of all other genetic loci are homozygous such that an organ, tissue, or cell, from one animal can be used to prolong acceptance in a recipient, e.g., a xenorecipient, of an organ, tissue, or cell, from a second animal from a herd of such animals, or such that prolongation of acceptance (e.g., by the induction of tolerance) in a recipient, e.g., a xenorecipient, of an organ, tissue, or cell, from one animal of the herd also provides prolongation of acceptance of an organ, tissue, or cell, from a second animal of the herd.
Accordingly, the invention features, a swine, preferably a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous. In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In preferred embodiments, the swine is capable of reproduction, i.e., the animal can produce functional gametes.
In another aspect, the invention features, a cell or a preparation of such cells, from a swine, preferably a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
In preferred embodiments, the swine cell is an embryonic stem cell. In other preferred embodiments, the swine cell can be a hematopoietic stem cell, e.g., a cord blood hematopoietic stem cell, a bone marrow hematopoietic stem cell, or a fetal or neonatal liver or spleen hematopoietic stem cell; a differentiated blood cell, e.g., a myeloid cell, a megakaryocyte, a monocyte, a granulocyte, an eosinophil, an erythroid cell, a lymphoid cell, such as a B e o lymphocyte or a T lymphocyte; a pluripotent hematopoietic stem cell, e.g., a hematopoietic precursor, a burst-forming units-erythroid (BFU-E), a colony forming unit-erythroid (CFU-E), a colony forming unit-megakaryocyte (CFU-Meg), a colony forming unit-granulocyte-monocyte (CFU-GM), a colony forming unit-eosinophil (CFU-Eo), or a colony forming unit-granulocyte-erythrocyte-megakaryocyte-monocyte (CFU-GEMM); a swine cell other than a hematopoietic stem cell or other blood cell; a swine thymic cell, e.g., a swine thymic stromal cell; a bone marrow stromal cell; a swine liver cell; a swine kidney cell; a swine epithelial cell; a swine muscle cell, e.g., a heart cell; or a dendritic cell or precursor thereof.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine cell are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In another aspect, the invention features, an isolated cell nucleus from a swine cell, preferably a miniature swine cell, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous. In preferred embodiments, the cell nucleus is from an undifferentiated cell. In other embodiments, the cell nucleus is from a differentiated cell.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine cell nucleus are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In another aspect, the invention features, an isolated organ, or a tissue, from a swine, preferably a miniature swine, which swine is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
In preferred embodiments, the organ can be an organ of the gastrointestinal tract, a liver, a kidney, a pancreas, a stomach, a spleen, or a gallbladder; a sensory organ, e.g., an eye; a lung; on organ or tissue of the circulatory system, e.g., a heart. In other preferred embodiments, the tissue can be connective tissue; epithelial tissue, e.g., skin; muscle tissue; osseous tissue; vascular tissue, e.g., a blood vessel; or occular tissue, e.g., lens tissue.
In preferred embodiments, the isolated organ or tissue is from a postnatal animal, e.g., a juvenile or adult animal, or a prenatal animal, e.g., a fetus or an embryo.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In another aspect, the invention features, a hematopoietic stem cell preparation, e.g., a bone marrow stem cell preparation, from a swine, preferably a miniature swine, which swine is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
In preferred embodiments, the hematopoietic stem cell preparation is from a postnatal animal, e.g., a juvenile or adult animal, or a prenatal animal, e.g., a fetus or an embryo.
In preferred embodiments, the preparation includes hematopoietic stem cells from cord blood, the liver, or spleen.
In preferred embodiments, the preparation is a bone marrow preparation which includes immature bone marrow cells, e.g., undifferentiated hematopoietic stem cells, in addition to other bone marrow components. In other preferred embodiments, the bone marrow preparation is composed of isolated undifferentiated hematopoietic stem cells.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In another aspect, the invention features, a herd of swine, preferably miniature swine, in which the animals are homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous. In preferred embodiments, the herd of swine includes at least one male swine and at least one female swine capable of reproduction, e.g., at least one male and one female which can produce functional gametes.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine in the herd are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In another aspect, the invention features, a method for providing a swine, preferably a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous. The method includes:
providing a first swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ but which is preferably homozygous at less than 20%, 30%. 50%, or 75% of all other loci;
(1) providing a second swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, which is of the same haplotype as the first swine, but which is preferably homozygous at less than 20%, 30%. 50%, or 75% of all other loci, which is preferably not a sibling, parent or offspring of the first swine;
(2) mating the first and second swine to provide an offspring;
(3) mating the offspring to a swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, which is of the same haplotype as the first swine but which is preferably homozygous at less than 20%, 30%. 50%, or 75% of all other loci, which is preferably not a sibling, parent or offspring of the offspring;
(4) repeating step (3) for at least 18 generations;
(5) performing a brother sister mating from the offspring of the final mating of step (4) to produce at least on male and one female sibling
(6) performing brother sister matings form the siblings of step (5) and for at least 5 additional generations,
to thereby provide a swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
In preferred embodiments, the swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, is mated in non brother-sister matings for at least 10, 15, 16, 17, 18, 19, 20, or 25 generations, and then mated in brother-sister matings for at least 4, 5, 6, 7, 8, 9, or 10 generations.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In preferred embodiments, the swine is capable of reproduction, i.e., the animal can produce functional gametes.
In another aspect, the invention features, a swine, preferably a miniature swine, made by a method described herein.
In another aspect, the invention features, a method of providing a swine, preferably a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous. The method includes mating a male swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous, with a female swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous, thereby providing a swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci of one or more of and more preferably all of the swine, the male swine, and the female swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj). In particularly preferred embodiments the halotype of swine, the male swine, and the female swine are the small.
In preferred embodiments, the swine is capable of reproduction, i.e., the animal can produce functional gametes.
In another aspect, the invention features, a method of providing a swine, preferably a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
The method includes: transferring swine genetic material, e.g., a cell nucleus or a set of chromosomes, e.g. a complete set of chromosomes, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ and in which at least 60% of all other genetic loci are homozygous, to a cell, wherein the cell is capable of developing into a swine, allowing the cell to develop into a swine, thereby providing a swine which is homozygous at swine leukocyte o antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
In preferred embodiments, the genetic material is transferred via nuclear transfer. For example, a swine cell nucleus, e.g., a nucleus from an undifferentiated swine cell, can be fused with a second cell, e.g., an oocyte, e.g., an enucleated oocyte, such as an enucleated oocyte arrested in the metaphase of the second meiotic division, and then transferred into a recipient swine, e.g., a maternal recipient swine. The embryo resulting from the fusion of the cell nucleus and the oocyte can also be cultured, e.g., cultured to the stage of blastocyst, and then transferred to the recipient swine.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In another aspect, the invention features, a method of providing a transgenic swine, e.g., a transgenic miniature swine. The method includes:
providing a swine, e.g., a miniature swine described herein, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which all other genetic loci are at least 60% homozygous; and
introducing a transgene into the swine, thereby preparing a transgenic swine.
In preferred embodiments the transgene encodes a xenogeneic, e.g., a human protein.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In preferred embodiments, the swine is capable of reproduction, i.e., the animal can produce functional gametes.
In another aspect, the invention features, a genetically engineered swine cell, e.g., a cultured swine cell, a retrovirally transformed swine cell, or a cell derived from a transgenic swine, or purified preparation of such cells, which include a transgene. The swine cell is from a swine, preferably a miniature swine, e.g., a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous.
In preferred embodiments the transgene encodes a xenogeneic, e.g., a human protein.
In yet other preferred embodiments the genetically engineered swine cell is: a swine hematopoietic stem cell, e.g., a cord blood hematopoietic stem cell, a bone marrow hematopoietic stem cell, or a fetal or neonatal liver or spleen hematopoietic stem cell; derived from differentiated blood cells, e.g. a myeloid cell, such as a megakaryocytes, monocytes, granulocytes, or an eosinophils; an erythroid cell, such as a red blood cells, e.g. a lymphoid cell, such as B lymphocytes and T lymphocytes; derived from a pluripotent hematopoietic stem cell, e.g. a hematopoietic precursor, e.g. a burst-forming units-erythroid (BFU-E), a colony forming unit-erythroid (CFU-E), a colony forming unit-megakaryocyte (CFU-Meg), a colony forming unit-granulocyte-monocyte (CFU-GM), a colony forming unit-eosinophil (CFU-Eo), or a colony forming unit-granulocyte-erythrocyte-megakaryocyte-monocyte (CFU-GEMM); a swine cell other than a hematopoietic stem cell, or other blood cell; a swine thymic cell, e.g., a swine thymic stromal cell; a bone marrow stromal cell; a swine liver cell; a swine kidney cell; a swine epithelial cell; a swine hematopoietic progenitor cell; a swine muscle cell, e.g., a heart cell; or a dendritic cell or precursor thereof.
In yet other preferred embodiments the transgenic cell is: isolated or derived from cultured cells, e.g., a primary culture, e.g., a primary cell culture of hematopoietic stem cells; isolated or derived from a transgenic animal.
In yet other preferred embodiments: the transgenic swine cell is hemizygous for the transgene; the transgenic swine cell is heterozygous for the transgene; the transgenic swine cell is homozygous for the transgene (heterozygous transgenic swine can be bred to produce offspring that are homozygous for the transgene); the transgenic swine cell includes two or more transgenes.
In another aspect, the invention features, a transgenic swine, e.g., a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous and having cells which include a transgene.
In preferred embodiments the transgene encodes a xenogeneic, e.g., a human protein.
In yet other preferred embodiments the transgene includes a nucleic acid encoding a human peptide, e.g., a hematopoietic peptide, operably linked to: a promoter other than the one it naturally occurs with; a swine promoter, e.g., a swine hematopoietic gene promoter; a viral promoter; or an inducible or developmentally regulated promoter.
In preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the transgenic swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In another aspect, the invention features, an isolated swine organ or a swine tissue from a transgenic swine, e.g., a miniature swine described herein, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous, having cells which include a xenogeneic, e.g., a human, nucleic acid.
In preferred embodiments the organ is a heart, lung, kidney, pancreas, or liver.
In preferred embodiments the tissue is: thymic tissue; islet cells or islets; stem cells; bone marrow; endothelial cells; skin; or vascular tissue.
In another aspect, the invention features, a method of inducing tolerance in a recipient mammal of a first species, e.g., a human, to a graft from a donor mammal of a second species, e.g., a swine, for example, a miniature swine described herein. The method includes:
providing a donor mammal, e.g., a miniature swine, which is from a herd which is homozygous for a major histocompatibility complex haplotype and at least 60% homozygous at all other genetic loci;
introducing into the recipient mammal, tolerance inducing tissue, e.g., hematopoietic stem cells from the donor mammal, thymic tissue from the donor mammal, or a nucleic acid which encodes an MHC antigen of the donor mammal;
providing a graft from the donor mammal or from a second donor mammal from the herd; and
introducing the graft into the recipient, thereby inducing tolerance in a recipient mammal of a first species to a graft from a mammal of the second species.
In another aspect, the invention features, a method of inducing tolerance in a recipient mammal of a first species, e.g., a human, to a graft from a donor mammal of a second species, e.g., a swine, for example, a miniature swine described herein. The method includes:
providing a donor mammal, e.g., a miniature swine, which is from a herd which is homozygous for a major histocompatibility complex haplotype and at least 60% homozygous at all other genetic loci;
introducing into the recipient mammal, hematopoietic stem cells from the donor mammal;
providing a graft from the donor mammal or from a second donor mammal from the herd; and
introducing the graft into the recipient, thereby inducing tolerance in a recipient mammal of a first species to a graft from a mammal of the second species.
In preferred embodiments, the recipient is a primate and the donor is a swine, e.g., a miniature swine; the recipient is a human and the donor is a swine, e.g., a miniature swine.
In preferred embodiments, the donor is a swine, preferably a miniature swine, which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and is from a herd which at least 60% of all other genetic loci are homozygous. In other preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In preferred embodiments the method is practiced without T cell depletion, e.g., without the administration of thymic irradiation, or T cell depleting anti T cell antibodies.
In preferred embodiments the method includes: administering to the recipient, one or both, of an inhibitor, e.g., a blocker, of the CD40 ligand-CD40 interaction and a blocker of the CD28-B7 interaction. The CD40 ligand-CD40 pathway can be inhibited by administering an antibody or soluble receptor for the CD40 ligand or CD40, e.g., by administering CTLA4-lgG. Preferably the inhibitor binds the CD40 ligand. The CD28-B7 pathway can be inhibited by administering a soluble receptor or antibody for the CD28 or B7, e.g., an anti-B7 antibody. Preferably, the inhibitor binds B7. In preferred embodiments CTLA4-lgG and an anti-b7 antibody are administered.
In preferred embodiments the method can be practiced without the administration of hematopoietic space-creating irradiation, e.g., whole body irradiation.
In preferred embodiments the method includes administering a sufficiently large number of donor hematopoietic cells to the recipient such that, donor stem cells engraft, give rise to mixed chimerism, and induce tolerance without space-creating treatment. In preferred embodiments the number of donor hematopoietic cells is at least twice, is at least equal to, or is at least 75, 50, or 25% as great as, the number of bone marrow cells found in an adult of the recipient species. In preferred embodiments the number of donor hematopoietic stem cells is at least twice, is at least equal to, or is at least 75, 50, or 25% as great as, the number of bone marrow hematopoietic stem cells found in an adult of the recipient species. In the case where an inbred population of the donor species exists, e.g., where the donor species is miniature swine, the number of available donor cells is not limited to the number of cells which can be obtained from a single animal. Thus, in such cases, the donor cells administered to the recipient can come from more than one, e.g., from two, three, four, or more animals.
The number of donor cells administered to the recipient can be increased by either increasing the number of stem cells provided in a particular administration or by providing repeated administrations of donor stem cells.
Repeated stem cell administration can promote engraftment, mixed chimerism, and long-term deletional tolerance in graft recipients. Thus, the invention also includes methods in which multiple hematopoietic stem cell administrations are provided to a recipient. Multiple administration can substantially reduce or eliminate the need for hematopoietic space-creating irradiation. Administrations can be given prior to, at the time of, or after graft implantation. In preferred embodiments multiple administrations of stem cells are provided prior to the implantation of a graft. Two, three, four, five, or more administrations can be provided. The period between administrations of hematopoietic stem cells can be varied. In preferred embodiments a subsequent administration of hematopoietic stem cell is provided: at least two days, one week, one month, or six months after the previous administration of stem cells; when the recipient begins to show signs of host lymphocyte response to donor antigen; when the level of chimerism decreases; when the level of chimerism falls below a predetermined value; when the level of chimerism reaches or falls below a level where staining with a monoclonal antibody specific for a donor PBMC antigen is equal to or falls below staining with an isotype control which does not bind to PBMC""s, e.g. when the donor specific monoclonal stains less than 1-2% of the cells; or generally, as is needed to maintain a level of mixed chimerism sufficient to maintain tolerance to donor antigen.
When multiple stem cell administrations are given, one or more of the administrations can include a number of donor hematopoietic cells which is at least twice, is equal to, or is at least 75, 50, or 25% as great as, the number of bone marrow cells found in an adult of the recipient species; include a number of donor hematopoietic stem cells which is at least twice, is equal to, or is at least 75, 50, or 25% as great as, the number of bone marrow hematopoietic stem cells found in an adult of the recipient species.
Although the methods described herein, e.g., those in which blockers of both pathways are administered, or those in which a relatively large number of hematopoietic stem cells are administered, will often eliminate the need for other preparative steps, some embodiments include inactivating preferably graft reactive or xenoreactive, e.g., swine reactive, NK cells, of the recipient mammal. This can be accomplished, e.g., by introducing into the recipient mammal an antibody capable of binding to natural killer cells of the recipient mammal. The administration of antibodies, or other treatment to inactivate natural killer cells, can be given prior to introducing the hematopoietic stem cells into the recipient mammal or prior to implanting the graft in the recipient. This antibody can be the same or different from an antibody used to inactivate T cells.
Although the methods described herein, e.g., those in which blockers of both pathways are administered, or those in which a relatively large number of hematopoietic stem cells are administered, will often eliminate the need for other preparative steps, some embodiments include inactivating e.g., by depleting natural killer cells, T cells, preferably graft reactive or xenoreactive, e.g., swine reactive, T cells of the recipient mammal. This can be accomplished, e.g., by introducing into the recipient mammal an antibody capable of binding to T cells of the recipient mammal. The administration of antibodies, or other treatment to inactivate T cells, can be given prior to introducing the hematopoietic stem cells into the recipient mammal or prior to implanting the graft in the recipient. This antibody can be the same or different from an antibody used to inactivate natural killer cells.
Other preferred embodiments include: the step of introducing into the recipient mammal, donor species-specific stromal tissue, preferably hematopoietic stromal tissue, e.g., fetal liver or thymus. In preferred embodiments: the stromal tissue is introduced simultaneously with, or prior to, the hematopoietic stem cells; the hematopoietic stem cells are introduced simultaneously with, or prior to, the antibody.
Although the methods described herein, e.g., those in which blockers of both pathways are administered, or those in which a relatively large number of hematopoietic stem cells are administered, will often eliminate the need for other preparative steps, some embodiments include (optionally): the step of, prior to hematopoietic stem cell transplantation, creating hematopoietic space, e.g., by irradiating the recipient mammal with low dose, e.g., less than 400, preferably less than 300, more preferably less than 200 or 100 rads, whole body irradiation to deplete or partially deplete the bone marrow of the recipient. As is discussed herein this treatment can be reduced or entirely eliminated.
Other preferred embodiments include: the step of, preferably prior to hematopoietic stem cell transplantation, depleting natural antibodies from the blood of the recipient mammal. Depletion can be achieved, by way of example, by contacting the recipients blood with an epitope which absorbs performed anti-donor antibody. The epitope can be coupled to an insoluble substrate and provided, e.g., as an affinity column. E.g., an xcex11-3 galactose linkage epitope-affinity matrix, e.g., matrix bound linear B type VI carbohydrate, can be used to deplete natural antibodies. Depletion can also be achieved by hemoperfusing an organ, e.g., a liver or a kidney, obtained from a mammal of the donor species. (In organ hemoperfusion antibodies in the blood bind to antigens on the cell surfaces of the organ and are thus removed from the blood).
Other preferred embodiments include those in which: the same mammal of the second species is the donor of one or both the graft and the hematopoietic cells.
In preferred embodiments, the method includes the step of introducing into the recipient a graft obtained from the donor which is obtained from a different organ than the hematopoietic stem cells, e.g., a heart, pancreas, liver, or kidney.
In preferred embodiments the host or recipient is a post-natal individual, e.g., an adult, or a child.
In preferred embodiments the method further includes the step of identifying a host or recipient which is in need of a graft.
In another aspect, the invention features a method of inducing tolerance in a recipient mammal of a first species, e.g., a human, to a graft from a donor mammal of a second species, e.g., a swine, for example, a miniature swine. The method includes:
providing a donor mammal which is from a herd which is homozygous for a major histocompatibility complex haplotype and at least 60% homozygous at all other genetic loci;
introducing into the recipient mammal, thymic tissue from the donor mammal;
providing a graft from the donor mammal, or from a second donor mammal from the herd; and
introducing the graft into the recipient, thereby inducing tolerance in a recipient mammal of a first species to a graft from a mammal of the second species.
In preferred embodiments, the recipient is a primate and the donor is a swine, e.g., a miniature swine; the recipient is a human and the donor is a swine, e.g., a miniature swine.
In preferred embodiments, the donor is a swine and is from a herd which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and is from a herd in which at least 60% of all other genetic loci are homozygous. In other preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine herd are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
In preferred embodiments the method is practiced without T cell depletion or inactivation, e.g., without the administration of thymic irradiation, or T cell depleting anti T cell antibodies.
In preferred embodiments the method includes: administering to the recipient, one or both, of an inhibitor, e.g., a blocker, of the CD40 ligand-CD40 interaction and a blocker of the CD28-B7 interaction. The CD40 ligand-CD40 pathway can be inhibited by administering an antibody or soluble receptor for the CD40 ligand or CD40, e.g., by administering CTLA4-lgG. Preferably the inhibitor binds the CD40 ligand. The CD28-B7 pathway can be inhibited by administering a soluble receptor or antibody for the CD28 or B7, e.g., an anti-B7 antibody. Preferably, the inhibitor binds B7. In preferred embodiments CTLA4-lgG and an anti-7 antibody are administered.
Although the methods described herein, e.g., those in which blockers of both pathways are administered, will often eliminate the need for other preparative steps, some embodiments include inactivating natural killer cells, preferably graft reactive or xenoreactive, e.g., swine reactive, NK cells, of the recipient mammal. This can be accomplished, e.g., by introducing into the recipient mammal an antibody capable of binding to natural killer cells of the recipient mammal. The administration of antibodies, or other treatment to inactivate natural killer cells, can be given prior to introducing the thymic tissue into the recipient mammal or prior to implanting the graft in the recipient. This antibody can be the same or different from an antibody used to inactivate T cells.
Although methods described herein, e.g., those in which blockers of both pathways are administered, will often eliminate the need for other preparative steps, some embodiments include inactivating, e.g., by depleting T cells, preferably graft reactive or xenoreactive, e.g., swine reactive, T cells of the recipient mammal. This can be accomplished, e.g., by introducing into the recipient mammal an antibody capable of binding to T cells of the recipient mammal. The administration of antibodies, or other treatment to inactivate T cells, can be given prior to introducing the thymic tissue into the recipient mammal or prior to implanting the graft in the recipient. This antibody can be the same or different from an antibody used to inactivate natural killer cells.
Other preferred embodiments include: the step of, preferably prior to thymic tissue transplantation, depleting natural antibodies from the blood of the recipient mammal. Depletion can be achieved, by way of example, by contacting the recipients blood with an epitope which absorbs performed anti-donor antibody. The epitope can be coupled to an insoluble substrate and provided, e.g., as an affinity column. E.g., an xcex11-3 galactose linkage epitope-affinity matrix, e.g., matrix bound linear B type VI carbohydrate, can be used to deplete natural antibodies. Depletion can also be achieved by hemoperfusing an organ, e.g., a liver or a kidney, obtained from a mammal of the donor species. (In organ hemoperfusion antibodies in the blood bind to antigens on the cell surfaces of the organ and are thus removed from the blood.)
Other preferred embodiments include those in which: the same mammal of the second species is the donor of one or both the graft and the thymic tissue.
In preferred embodiments, the method includes the step of introducing into the recipient a graft obtained from the donor which is obtained from a different organ than the thymic tissue, e.g., a heart, pancreas, liver, or kidney.
In preferred embodiments the host or recipient is a post-natal individual, e.g., an adult, or a child.
In preferred embodiments the method further includes the step of identifying a host or recipient which is in need of a graft.
In another aspect, the invention features a method of inducing tolerance in a recipient mammal, preferably a primate, e.g., a human, to a graft obtained from a donor mammal of a second species, e.g., a swine, e.g., a miniature swine, which graft preferably expresses an MHC antigen.
The method includes:
inserting a nucleic acid, e.g., DNA, encoding an MHC antigen into a hematopoietic stem cell, e.g., bone marrow hematopoietic stem cell, of the recipient, wherein the nucleic acid encodes an MHC antigen of a swine, e.g., a miniature swine, from a herd which is homozygous for a major histocompatibility complex haplotype and at least 60% homozygous at all other genetic loci;
allowing the MHC antigen encoding nucleic acid to be expressed in the recipient; and
preferably, implanting the graft in the recipient, wherein the graft is from an animal from the herd.
In preferred embodiments, the donor is a swine from a herd which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other do genetic loci are homozygous. In other preferred embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, of all other genetic loci in the swine are homozygous.
In preferred embodiments, the swine leukocyte antigens (SLA) A, B, C, DR, and DQ can be of haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc), haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), or haplotype j (Aj, Bj, Cj, DRj, DQj).
Preferred embodiments include those in which: the cell is removed from the recipient prior to the nucleic acid insertion and returned to the recipient after the nucleic acid insertion; the nucleic acid includes a MHC class I gene, e.g., a (SLA) A, B, C gene; the nucleic acid includes a MHC class II gene, e.g., a DR or DQ gene; the nucleic acid is inserted into the cell by transduction, e.g. by a retrovirus, e.g., by a Moloney-based retrovirus; and the nucleic acid is expressed in bone marrow cells and/or peripheral blood cells of the recipient at least 14, preferably 30, more preferably 60, and most preferably 120 days, after the nucleic acid is introduced into the recipient.
In preferred embodiments the method is practiced without T cell depletion, e.g., without the administration of thymic irradiation, or T cell depleting anti T cell antibodies.
In preferred embodiments the method includes: administering to the recipient, one or both, of an inhibitor, e.g., a blocker, of the CD40 ligand-CD40 interaction and a blocker of the CD28-B7 interaction. The CD40 ligand-CD40 pathway can be inhibited by administering an antibody or soluble receptor for the CD40 ligand or CD40, e.g., by administering CTLA4-lgG. Preferably the inhibitor binds the CD40 ligand. The CD28-B7 pathway can be inhibited by administering a soluble receptor or antibody for the CD28 or B7, e.g., an anti-B7 antibody. Preferably, the inhibitor binds B7. In preferred embodiments CTLA4-lgG and an anti-7 antibody are administered.
Although the methods described herein, e.g., those in which blockers of both pathways are administered, will often eliminate the need for other preparative steps, some embodiments include inactivating natural killer cells, preferably graft reactive or xenoreactive, e.g., swine reactive, NK cells, of the recipient mammal. This can be accomplished, e.g., by introducing into the recipient mammal an antibody capable of binding to natural killer cells of the recipient mammal. The administration of antibodies, or other treatment to inactivate natural killer cells, can be given prior to introducing the hematopoietic stem cells into the recipient mammal or prior to implanting the graft in the recipient. This antibody can be the same or different from an antibody used to inactivate T cells.
Although the methods described herein, e.g., those in which blockers of both pathways are administered, will often eliminate the need for other preparative steps, some embodiments include inactivating T cells, preferably graft reactive or xenoreactive, e.g., swine reactive, T cells of the recipient mammal. This can be accomplished, e.g., by introducing into the recipient mammal an antibody capable of binding to T cells of the recipient mammal. The administration of antibodies, or other treatment to inactivate, e.g., deplete, T cells, can be given prior to introducing the hematopoietic stem cells into the recipient mammal or prior to implanting the graft in the recipient. This antibody can be the same or different from an antibody used to inactivate natural killer cells.
Preferred embodiments include (optionally): the step of, prior to engineered hematopoietic stem cell transplantation, creating hematopoietic space, e.g., by irradiating the recipient mammal with low dose, e.g., less than 400, preferably less than 300, more preferably less than 200 or 100 rads, whole body irradiation to deplete or partially deplete the bone marrow of the recipient.
Other preferred embodiments include: the step of, preferably prior to engineered hematopoietic stem cell transplantation, depleting natural antibodies from the blood of the recipient mammal. Depletion can be achieved, by way of example, by contacting the recipients blood with an epitope which absorbs performed anti-donor antibody. The epitope can be coupled to an insoluble substrate and provided, e.g., as an affinity column. E.g., an xcex11-3 galactose linkage epitope-affinity matrix, e.g., matrix bound linear B type VI carbohydrate, can be used to deplete natural antibodies. Depletion can also be achieved by hemoperfusing an organ, e.g., a liver or a kidney, obtained from a mammal of the donor species. (In organ hemoperfusion antibodies in the blood bind to antigens on the cell surfaces of the organ and are thus removed from the blood.)
In preferred embodiments, the method includes the step of introducing into the recipient a graft obtained from the donor which is obtained from a different organ than the hematopoietic stem cells, e.g., a heart, pancreas, liver, or kidney.
In preferred embodiments the host or recipient is a post-natal individual, e.g., an adult, or a child.
In preferred embodiments the method further includes the step of identifying a host or recipient which is in need of a graft.
The retroviral methods of the invention allow the reconstitution of a graft recipient""s bone marrow with transgenic autologous bone marrow cells expressing a donor MHC gene. Expression of a transgenic MHC gene confers tolerance to grafts which exhibit the products of these or closely related MHC genes. Thus, these methods provide for the induction of specific transplantation tolerance by somatic transfer of MHC genes. Retroviral methods of the invention avoid the undesirable side effects of broad spectrum immune suppressants which are often used in transplantation.
In another aspect, the invention features, a method of selectively breeding animals described herein to improve or maintain fecundity of a herd. The method includes:
mating a first sow of a herd with a mate from the herd;
mating a second sow of the herd with the same or a different male from the herd;
determining which sow has higher fecundity;
mating the sow with the highest fecundity (or an offspring of said sow) to thereby improve or maintain fecundity of the herd.
A herd of the invention can be expanded by matings between males and females drawn from the herd. The zygotes which result from such matings can be allowed to develop in the female which produced the egg or eggs which were fertilized in the mating. The herd can also be expanded by implanting a zygote (wherein the zygote produced by the union of a sperm cell produced by a male of the herd with an egg produced by a female of the herd) in a foster mother. The foster mother can be from the herd or can be an animal which is not from the herd. For example, a xe2x80x9cherdxe2x80x9d zygote can be implanted in an outbred foster mother. This method can allow for rapid expansion of a herd. Accordingly, in another aspect, the invention features a method of expanding an inbred herd, e.g., a herd described herein. The method includes:
providing a zygote which is produced by the union of a sperm cell produced by a male of the herd with an egg produced by a female of the herd;
implanting the zygote into a foster mother, e.g., a female which is preferably not from the herd, allowing the zygote to give rise to an inbred swine, thereby expanding the herd.
xe2x80x9cA preparation of cellsxe2x80x9d, as used herein, refers to cells which are physically separated from the animal which produces them.
xe2x80x9cAn isolated nucleusxe2x80x9d, as used herein, refers to a nucleus which has been removed from the cell of its origin.
xe2x80x9cAn isolated organxe2x80x9d, as used herein, refers to an organ or tissue which has been physically separated from the animal which produces it.
xe2x80x9cA hematopoietic stem cell preparation, as used herein, is a population of cells which includes hematopoietic stem cells. The preparation can be pure, or it can include other cell types.
xe2x80x9cA juvenile miniature swine, is a swine which has not reached sexual maturity.
xe2x80x9cAn adult miniature swinexe2x80x9d is one which has reached sexual maturity.
xe2x80x9cA herd,xe2x80x9d as used herein, refers to a group of at least one male and one female which can breed to produce fertile male and female offspring. All of the animals of a herd are homozygous at SLA loci: A, B, C, DR and DQ, and all animals in the herd are homozygous for the same allele at SLA A, B, C, DR and DQ. Thus, only one allele for each of SLA A, B, C, DR, or DQ is present in the herd. Furthermore, the herd is highly inbred at all other loci. At least 60%, and preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, of all other loci are homozygous and for each of their loci, all swine in the herd are homozygous for the same allele. Thus in a herd wherever at least 85% of the loci are homozygous, there is no genetic variation in the herd for at least 85% of the loci. Homozygosity can be determined, e.g., by minisatellite analysis or mathematically.
xe2x80x9cGraftxe2x80x9d, as used herein, refers to a body part, organ, tissue, or cells. Organs such as liver, kidney, heart or lung, or other body parts, such as bone or skeletal matrix, tissue, such as skin, intestines, endocrine glands, or progenitor stem cells of various types, are all examples of grafts.
xe2x80x9cHematopoietic stem cellxe2x80x9d, as used herein, refers to a cell, e.g., a bone marrow cell, or a fetal liver or spleen cell, which is capable of developing into all myeloid and lymphoid lineages and by virtue of being able to self-renew can provide long term hematopoietic reconstitution. Preparations of hematopoietic cells or preparations, such as bone marrow, which include other cell types, can be used in methods of the invention. Although not wishing to be bound by theory, it is believed that the hematopoietic stem cells home to a site in the recipient mammal. The preparation should include immature cells, i.e., undifferentiated hematopoietic stem cells; these desired cells can be separated out of a preparation or a complex preparation can be administered. E.g., in the case of bone marrow stem cells, the desired primitive cells can be separated out of a preparation or a complex bone marrow sample including such cells can be used. Hematopoietic stem cells can be from fetal, neonatal, immature or mature animals. Stem cells derived from the cord blood of the recipient or the donor can be used in methods of the invention. See U.S. Pat. No. 5,192,553, hereby incorporated by reference, and U.S. Pat. No. 5,004,681, hereby incorporated by reference.
xe2x80x9cThymic or lymph node or thymocytes or T cellxe2x80x9d, as used herein, refers to thymocytes or T cells which are resistant to inactivation by traditional methods of T cell inactivation, e.g., inactivation by a single intravenous administration of anti-T cell antibodies, e.g., antibodies, e.g., ATG preparation.
xe2x80x9cThymic irradiationxe2x80x9d, as used herein, refers to a treatment in which at least half, and preferably at least 75, 90, or 95% of the administered irradiation is targeted to the thymus. Whole body irradiation, even if the thymus is irradiated in the process of delivering the whole body irradiation, is not considered thymic irradiation.
xe2x80x9cMHC antigenxe2x80x9d, as used herein, refers to a protein product of one or more MHC genes; the term includes fragments or analogs of products of MHC genes which can evoke an immune response in a recipient organism. Examples of MHC antigens include the products (and fragments or analogs thereof) of the human MHC genes, i.e., the HLA genes. MHC antigens in swine, e.g., miniature swine, include the products (and fragments and analogs thereof) of the SLA genes, e.g., the DRB gene.
xe2x80x9cHematopoietic space-creating irradiationxe2x80x9d, as used herein, refers to irradiation directed to the hematopoietic tissue, i.e., to tissue in which stem cells are found, e.g., the bone marrow. It is of sufficient intensity to kill or inactivate a substantial number of hematopoietic cells. It is often given as whole body irradiation.
xe2x80x9cThymic spacexe2x80x9d as used herein, is a state created by a treatment that facilitates the migration to and/or development in the thymus of donor hematopoietic cells of a type which can delete or inactivate host thymocytes that recognize donor antigens. It is believed that the effect is mediated by elimination of host cells in the thymus.
xe2x80x9cStromal tissuexe2x80x9d, as used herein, refers to the supporting tissue or matrix of an organ, as distinguished from its functional elements or parenchyma.
xe2x80x9cTolerancexe2x80x9d, as used herein, refers to an inhibition of a graft recipient""s immune response which would otherwise occur, e.g., in response to the introduction of a non-self MHC antigen into the recipient. Tolerance can involve humoral, cellular, or both humoral and cellular responses. Tolerance, as used herein, refers not only to complete immunologic tolerance to an antigen, but to partial immunologic tolerance, i.e., a degree of tolerance to an antigen which is greater than what would be seen if a method of the invention were not employed. Tolerance, as used herein, refers to a donor antigen-specific inhibition of the immune system as opposed to the broad spectrum inhibition of the immune system seen with immunosuppressants.
xe2x80x9cA blockerxe2x80x9d as used herein, refers to a molecule which binds a member of a ligand/counter-ligand pair and inhibits the interaction between the ligand and counter-ligand or which disrupts the ability of the bound member to transduce a signal. The blocker can be an antibody (or fragment thereof) to the ligand or counter ligand, a soluble ligand (soluble fragment of the counter ligand), a soluble counter ligand (soluble fragment of the counter ligand), or other protein, peptide or other molecule which binds specifically to the counter-ligand or ligand, e.g., a protein or peptide selected by virtue of its ability to bind the ligand or counter ligand in an affinity assay, e.g., a page display system.
The term xe2x80x9chaplotypexe2x80x9d as used herein refers to a group of alleles from closely linked loci which are usually inherited as a unit. For example, in the MHC locus in swine the SLAa haplotype codes for the SLA-Aa, Ba, Ca, DRa, and DQa alleles, the SLAd haplotype codes for the SLA-Ad, Bd, Cd, DRd, and DQd alleles, etc.
The terms xe2x80x9corganxe2x80x9d and xe2x80x9ctissuexe2x80x9d as used herein, mean any biological material that is capable of being transplanted and include organs (especially the internal vital organs such as the heart, lung, liver, kidney, pancreas and thyroid), cornea, skin, blood vessels and other connective tissue, cells including blood and hematopoietic cells, Islets of Langerhans, brain cells and cells from endocrine and other organs and bodily fluids, all of which may be candidate for transplantation.
As used herein, the term xe2x80x9ctransgenexe2x80x9d refers to a nucleic acid sequence (encoding, e.g., one or more class I or class II MHC proteins), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced or which when introduced into the genome results in a change of sequence in the genome. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, a xe2x80x9ctransgenic swinexe2x80x9d is any swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous, and in which one or more, and preferably essentially all, of the cells of the animal include a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9cgenetically engineered swine cellsxe2x80x9d refers to cells derived from a swine which is homozygous at swine leukocyte antigens (SLA) A, B, C, DR, and DQ, and in which at least 60% of all other genetic loci are homozygous, and which have been used as recipients for a recombinant vector or other transfer nucleic acid, and include the progeny of the original cell which has been transfected or transformed. Genetically engineered swine cells include cells in which transgenes or other nucleic acid vectors have been incorporated into the host cell""s genome, as well as cells harboring expression vectors which remain autonomous from the host cell""s genome.
As used herein, the term xe2x80x9cpropagatablexe2x80x9d refers to animals which are capable of giving rise to viable offspring by sexual or asexual reproduction. Preferably, animals of the invention are propagatable.
The high degree of genetic uniformity characteristic of animals described herein allows for considerable advantages in terms of quality assurance. For example, any single animal is representative of the herd, i.e., the same or very similar (allow for differences of are or gender) to any other, in terms of immunogenetics, size, physiology, and health.
Genetically uniform animals described herein are useful genetic engineering. for example, a first modification, e.g., the introduction of a first transgene can be made in a first animal. A second modification, e.g., the introduction of a different second transgene, can be made in a second animal. The appropriate matings can be performed to yield an animal having both modifications. Except for the modifications, all of the modified animals, as well as non-modified animals of the herd, are highly uniform. Thus, genetically engineered modifications can be introduced by matings between modified animals, with minimal introduction of changes in the genetic background.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The drawings are first briefly described.